Prelimbic cortical pyramidal neurons to ventral tegmental area projections promotes arousal from sevoflurane anesthesia

Abstract Aims General anesthesia has been used in surgical procedures for approximately 180 years, yet the precise mechanism of anesthetic drugs remains elusive. There is significant anatomical connectivity between the ventral tegmental area (VTA) and the prelimbic cortex (PrL). Projections from VTA dopaminergic neurons (VTADA) to the PrL play a role in the transition from sevoflurane anesthesia to arousal. It is still uncertain whether the prelimbic cortex pyramidal neuron (PrLPyr) and its projections to VTA (PrLPyr‐VTA) are involved in anesthesia‐arousal regulation. Methods We employed chemogenetics and optogenetics to selectively manipulate neuronal activity in the PrLPyr‐VTA pathway. Electroencephalography spectra and burst‐suppression ratios (BSR) were used to assess the depth of anesthesia. Furthermore, the loss or recovery of the righting reflex was monitored to indicate the induction or emergence time of general anesthesia. To elucidate the receptor mechanisms in the PrLPyr‐VTA projection's impact on anesthesia and arousal, we microinjected NMDA receptor antagonists (MK‐801) or AMPA receptor antagonists (NBQX) into the VTA. Results Our findings show that chemogenetic or optogenetic activation of PrLPyr neurons prolonged anesthesia induction and promoted emergence. Additionally, chemogenetic activation of the PrLPyr‐VTA neural pathway delayed anesthesia induction and promoted anesthesia emergence. Likewise, optogenetic activation of the PrLPyr‐VTA projections extended the induction time and facilitated emergence from sevoflurane anesthesia. Moreover, antagonizing NMDA receptors in the VTA attenuates the delayed anesthesia induction and promotes emergence caused by activating the PrLPyr‐VTA projections. Conclusion This study demonstrates that PrLPyr neurons and their projections to the VTA are involved in facilitating emergence from sevoflurane anesthesia, with the PrLPyr‐VTA pathway exerting its effects through the activation of NMDA receptors within the VTA.


| INTRODUC TI ON
Nearly 180 years have elapsed since the initial use of general anesthesia in surgical procedures.However, the exact mechanism of general anesthesia remains incompletely understood.2][3] Moreover, investigating the mechanisms of general anesthesia enhances our understanding of consciousness in neuroscience research, with the study of neural circuits playing a pivotal role in decoding these mechanisms.
Research suggests that the central nervous system regulates the transition between anesthesia-induced unconsciousness and arousal through intricate neural networks. 46][7] However, understanding of the anesthetic drugs' mechanisms, based solely on the ascending pathways projecting to the cortex is limited. 8Recent advances in cortical research techniques have highlighted the role of intra-cortical and cortico-subcortical projections in managing the loss and recovery of consciousness under anesthesia. 9,10Nonetheless, these studies primarily focus on disruptions in cortical-cortical and cortical-thalamic information transmission, 11,12 as well as alterations in consciousness due to the loss of cortical information integration. 13,14The role of descending neural pathways, particularly from the cortex to subcortical nuclei, in the loss and recovery of consciousness remains largely unexplored.
Unlike most cortical regions, the PFC not only receives inputs but also projects them to downstream cell groups, exerting descending control over ascending regulatory systems. 15The medial prefrontal cortex (mPFC) is a crucial region for mediating higher cognitive functions and regulating anesthesia-induced consciousness. 15It has extensive neural projections to several arousal-promoting nuclei in the subcortex. 16,172][23] Previous research has highlighted the significance of the VTA-PrL neural pathway in the transition from sevoflurane anesthesia to arousal. 24Furthermore, studies report that pyramidal neurons in PrL can modulate VTA dopamine neuron activity, and inhibiting PrL Pyr neuron excitatory transmission can reduce morphine-induced dopamine neuronal activity enhancement in the VTA. 25 However, the role of PrL Pyr neurons and the PrL Pyr -VTA pathway in regulating anesthesia and arousal remains unclear.
In this study, we selectively activated PrL Pyr neurons and their projections to the VTA by employing transgenic viral transduction, chemogenetics, optogenetics, and pharmacological methods.
We investigated the role of PrL Pyr neurons and the PrL Pyr

| Stereotaxic surgery
For experimental preparation, the mouse received an intraperitoneal injection of 2% pentobarbital sodium (40-50 mg/kg) for general anesthesia and local anesthesia with 1% lidocaine to the head.Subsequently, the head was immobilized with a stereotaxic apparatus (RWD, Shenzhen, China).Body temperature was maintained at 35-36°C using a heating pad.To validate the function of PrL Pyr neurons, an adeno-associated viral vector was injected into the PrL at a rate of 30 nL/min using a microinjection pump (Harvard Apparatus, USA).The activation experiment involved unilateral injection, while the inhibition experiment entailed bilateral injection.Stereotaxic reference atlas, 26 with coordinates referenced to the Bregma.The

| Anesthesia induction and recovery time calculation
The righting reflex barrel, equipped with an anesthesia gas monitor connected through a circular hole on top (oxygen flow rate: 2 L/min, oxygen concentration: 40%), was used.Initially, mice were allowed to move freely inside the barrel for 15 min.Subsequently, 2.0% sevoflurane inhalation anesthesia was administered.The righting reflex was evaluated by rotating the barrel 180 degrees every 15 s following the induction of anesthesia.If the mouse remained in a dorsally recumbent position (limbs up) and could not spontaneously return to an upright posture, the loss of the righting reflex (LORR) was considered to have occurred, and the time was recorded as the anesthesia induction time.After LORR, sevoflurane anesthesia was maintained by continuous inhalation for 30 min.The vaporizer was then turned off, and the oxygen concentration was maintained while monitoring the righting reflex.The barrel was rotated 180 degrees every 15 s, and if the mouse spontaneously returned to an upright posture (limbs touching the ground), the recovery of the righting reflex (RORR) was considered to have occurred.Anesthesia recovery time was measured from the end of anesthesia to RORR.

| EEG recording
Five days after completing the righting reflex experiment, the mice were placed in the barrel and connected to EEG electrodes for monitoring.EEG signals were recorded using a signal amplification system (AD Instruments) and LabChart software (Analog Devices, USA) at a sampling rate of 1000 Hz.For optogenetic EEG activity monitoring, mice were acclimatized in the righting reflex barrel for 15 min.Subsequently, a 5-min awake baseline was recorded, followed by 30 min of 2.0% sevoflurane inhalation anesthesia, 2 min of photo stimulation occurred during the 25-27 min of the process.Data analysis was conducted using MATLAB (R2020a; MathWorks, USA).Data containing artifacts was excluded, and a filter was employed to eliminate 50 Hz power line interference.
Data within the 0.3-50 Hz frequency range were extracted for analysis, focusing specifically on the burst-suppression ratio before, during, and after the photo stimulation session.For chemogenetic EEG activity monitoring, mice were acclimatized in the righting reflex barrel for 15 min.This was followed by a 5-min awake baseline recording and 30 min of 2.0% sevoflurane inhalation anesthesia.The burst-suppression ratio was analyzed for the 5 min before and after the termination of anesthetic delivery.
Burst suppression ratio BSR, total power percentage analysis, and spectrum drawing were completed in MATLAB and based on our team's previous studies. 27,28

| Optogenetic technique
For the validation of PrL Pyr or PrL Pyr -VTA projection function, an optogenetic virus (rAAV2/9-CaMKII-ChR2-mCherry-WPRE-pA) or a control virus (rAAV2/9-CaMKII-mCherry-WPRE-pA) was injected into the PrL region.For the validation of PrL Pyr function, a ceramic-inserted fiber was positioned 100 μm above the virus injection site.For the validation of the PrL Pyr -VTA projections, the virus was injected into the PrL region, and a ceramic-inserted fiber was inserted into the VTA region.Mice were tested 4 weeks after viral transduction and expression.Before photo stimulation, the mice were allowed to acclimatize in the righting reflex barrel for 15 min, followed by 2.0% sevoflurane inhalation anesthesia for 30 min, and simultaneous blue photo stimulation with a 473-nm laser (5 mW, 20 Hz, 10 ms duration, 1 s on/1 s off cycle) was applied.Anesthesia induction time was documented.Five days later, the mice were tested for anesthesia recovery time.Mice were anesthetized with 2% sevoflurane for 30 min, after which the vaporizer was turned off and photo stimulation was applied to document the anesthesia recovery time.

| Immunofluorescence staining
Following the completion of behavioral experiments, including righting reflex and EEG monitoring, mice were deeply anesthetized using sevoflurane and subsequently perfused with 4% paraformaldehyde (PFA), followed by 0.9% saline.The brain slices were washed three times (5 min each) with 1× PBS to remove OCT.Subsequently, they were incubated with BSA at room temperature for 30 min.After that, the diluted primary antibodies, including glutamate antibody (1:1000, Sigma, G6642, rabbit) and Anti-c-fos antibody (1:1000, Abcam, ab208942, mouse), were applied and incubated overnight at 4°C.
The slices underwent three 5-min washes with 1× PBS to remove the primary antibodies.Secondary antibodies (1:500, labeled with Alexa Fluor 488 goat anti-mouse IgG, Alexa Fluor 488 goat anti-rabbit IgG, Cy3-labeled donkey anti-mouse IgG, Cy3-labeled donkey anti-rabbit IgG from Shanghai Beyotime Biotechnology Co., Ltd.) were added and incubated in the dark at room temperature for 2 h.After another wash with 1× PBS, the slices were covered with DAPI and incubated for 15 min.Finally, the images were captured using a fluorescence microscope (Revolution, Echo, America).Experimenters conducting the microscopic analysis were blinded to the group assignments.

| Statistical analysis
We used GraphPad Prism 8.0 software (GraphPad, USA) and MATLAB (Mathwork, US) for statistical analysis and graphing.
Sample size was referenced from related literature in the field of this research, with 6-8 mice in each group. 28,29Data were presented as mean ± standard deviation.The normality of data distributions was tested by the Shapiro-Wilk test.Two-tailed Student's t-tests were used for comparing two groups, including righting reflex and immunostaining.Multiple t-tests were used for EEG spectrum analysis.
One-way analysis of variance (ANOVA) followed by Bonferroni's multiple comparison tests was used for pharmacological experiments.A two-way ANOVA followed by post-hoc Tukey's multiple comparison tests, was used for measuring BSR per minute during anesthesia.Statistical significance levels are indicated as follows: *p < 0.05, **p < 0.01, and ***p < 0.001.

| Chemogenetic activation of PrL Pyr neurons prolongs anesthesia induction and promotes anesthesia emergence
Adeno-associated viruses were microinjected into the PrL region to selectively manipulate PrL Pyr neurons (Figure 1A, left).Four weeks post-virus injection, to further investigate the effects of activating PrL Pyr neurons on sevoflurane anesthesia induction and emergence, mice were intraperitoneally injected with CNO.One hour later, the righting reflex test or EEG monitoring was conducted (Figure 1C).
Compared to the mCherry group, the hM3Dq group exhibited a significantly prolonged anesthesia induction time and a significantly shortened emergence time (Figure 1F, p < 0.001).EEG analysis revealed a significantly reduced burst suppression ratio in the hM3Dq group compared to the mCherry group (Figure 1G,H, p < 0.001).During the LORR process, EEG spectral analysis showed that the percentages of power in the δ bands decreased in the hM3Dq group (p < 0.001), while those in the α and β bands increased in the hM3Dq group (α, p < 0.001; β, p = 0.0033, Figure 1I, left).During the RORR process, the percentages of power in the δ bands decreased in the hM3Dq group (p < 0.001), while those in the α and β bands increased in the hM3Dq group (α, p = 0.0016; β, p < 0.001, Figure 1I, right).Following all behavioral experiments, and 1 h after the intraperitoneal administration of CNO, the mice were euthanized, and their brain sections underwent immunostaining; widespread mCherry fluorescence was observed in the PrL region (Figure 1A, right), indicating successful viral expression in pyramidal neurons (Figure 1B).C-fos immunofluorescence staining was performed on PrL region brain sections (Figure 1D).The results showed a significant increase in c-fos expression in the PrL region of the hM3Dq group compared to the mCherry group (Figure 1E, p < 0.001, Table S1).This indicates that the activity of PrL Pyr neurons was enhanced in the hM3Dq group.These behaviors and EEG findings suggest that the induction and emergence processes of sevoflurane anesthesia are regulated by PrL Pyr neurons.

| Optogenetic activation of PrL Pyr neurons delays anesthesia induction and promotes anesthesia emergence
We performed microinjections of adeno-associated viruses expressing ChR2 into the PrL region to selectively manipulate the activity of PrL Pyr neurons (Figure 2A, left).Four weeks post-virus injection, to investigate the effects of optogenetic activation of PrL Pyr neurons on induction and emergence from sevoflurane anesthesia, mice were connected to an optical fiber, placed in a righting reflex barrel, and underwent the righting reflex test or EEG monitoring (Figure 2C).Compared to the mCherry group, the induction time was prolonged, and the emergence time was significantly shortened in the ChR2 group (Figure 2D, p < 0.001).EEG monitoring revealed a significant reduction in the burst suppression ratio within 2 min of photo stimulation in the ChR2 group compared to the mCherry group (Figure 2G,H, p < 0.001).After EEG monitoring, mice were euthanized and subjected to immunofluorescence staining; widespread mCherry fluorescence was observed in the PrL region (Figure 2A, right), indicating successful viral expression in pyramidal neurons (Figure 2B).C-fos immunofluorescence staining was performed on PrL region brain sections (Figure 2E).The results showed a significant increase in c-fos expression in the PrL region of the ChR2 group compared to the mCherry group (Figure 2F, p < 0.001, Table S2).This indicates that the activity of PrL Pyr neurons was enhanced in the ChR2 group.These results suggest that the induction and emergence processes of sevoflurane anesthesia are regulated by PrL Pyr neurons.

| Chemogenetic activation of the PrL Pyr -VTA neural pathway delays anesthesia induction and promotes anesthesia emergence
The PrL region was targeted with injections of adeno-associated viruses expressing either the hM3Dq receptor or control viruses.
Additionally, retrograde viruses (rAAV2/Retro-CaMKII-NLS-Cre) were injected into the VTA region.This approach allowed for the selective manipulation of the PrL Pyr -VTA neural pathway (Figure 3A, left).Four weeks after virus injection, to further investigate the effects of activating the PrL Pyr -VTA neural pathway on sevoflurane anesthesia induction and emergence, mice were intraperitoneally injected with CNO.After 1 h, the righting reflex test or EEG monitoring was conducted (Figure 3B).Compared to the mCherry group, the hM3Dq group exhibited a significantly prolonged anes- EEG spectral analysis showed that after PrL Pyr -VTA was activated, the power ratio of the low-frequency band associated with anesthesia decreased, while the power ratio of the high-frequency band associated with wakefulness increased.After completion of all behavioral experiments, mice were euthanized, and subjected to immunofluorescence staining, widespread mCherry fluorescence was observed in the PrL region (Figure 3A, right), indicating successful viral expression in pyramidal neurons (Figure 3C).C-fos immunofluorescence staining was performed on brain sections in the PrL region (Figure 3D).The results showed a significant increase in c-fos expression in the PrL region of the hM3Dq group compared to the mCherry group (Figure 3E, p < 0.001, Table S3).Concurrently, we inhibited this pathway via chemogenetics, which can promote anesthesia induction and delay anesthesia emergence (see Figure S1).
These behaviors and EEG findings suggest that the induction and emergence processes of sevoflurane anesthesia are regulated by the PrL Pyr -VTA neural pathway.

| Optogenetic activation of the PrL Pyr -VTA neural pathway delays anesthesia induction and promotes anesthesia emergence
The PrL region received injections of adeno-associated viruses expressing ChR2 or control viruses, while a ceramic implant was surgically placed in the VTA region.This combination allowed for the selective manipulation of the PrL Pyr -VTA neural pathway (Figure 4A).
Optogenetic methods were employed to activate the PrL Pyr -VTA neural pathway and observe its effects on sevoflurane anesthesia induction and emergence.Mice were connected to an optical fiber and placed in a righting reflex barrel (Figure 4B).Compared to the mCherry group, the ChR2 group exhibited a significantly prolonged anesthesia induction time (Figure 4D, left, p = 0.0096) and a significantly shortened emergence time (Figure 4D, right, p = 0.0043).EEG monitoring showed a significantly reduced burst suppression ratio in the ChR2 group during photo stimulation compared to the mCherry group (Figure 4G,H, p < 0.001).After completion of all behavioral experiments, mice were euthanized, and the ceramic implant was found to be in the proper position (Figure 4C).Immunofluorescence staining was performed on brain sections in the VTA region (Figure 4E), and the results showed a significant increase in c-fos expression in the VTA region of the ChR2 group compared to the mCherry group (p < 0.001; Figure 4F, Table S4).These behaviors and immunofluorescence staining suggest that the PrL Pyr -VTA neural pathway can regulate the induction and emergence processes of sevoflurane anesthesia by exciting dopaminergic neurons in the VTA region.

| Chemogenetic inhibition of the PrL Pyr -VTA neural pathway promotes anesthesia induction and delays anesthesia emergence
The PrL region was targeted with injections of either the hM4Di re-

| DISCUSS ION
In this study, we combined chemogenetics and optogenetics with behavioral and EEG monitoring; this approach validated that activating PrL Pyr not only delays the induction of sevoflurane anesthesia but also promotes anesthesia arousal.Additionally, we applied the same methods to confirm that activating the PrL Pyr -VTA neural pathway can slow down the induction of sevoflurane anesthesia and accelerate anesthesia emergence.By employing chemical genetics combined with pharmacology, we demonstrated that the PrL Pyr -VTA neural pathway exerts its effects on promoting anesthesia emergence by acting on NMDA receptors in the VTA region.
Advancements in cortical observation techniques and intracortical electrode recordings have accumulated substantial knowledge about the effects of general anesthesia on the cortex.Another study has highlighted the "fragmentation" of information transmission between cortical regions as a significant factor leading to the loss of consciousness during general anesthesia. 11Anesthetics disrupt functional connectivity between cortical regions, impairing the cortex's ability to transmit and integrate information, resulting in the loss of consciousness. 14,30Researchers increasingly recognize the importance of the "Top-Down" mechanism in regulating the loss and recovery of consciousness during general anesthesia, but the cellular and circuit-level mechanisms underlying this remain unclear.
General anesthetics exhibit varying degrees of inhibition in different regions of the cerebral cortex, with a preference for inhibiting neurons in higher-order cortical regions while relatively preserving neurons in lower-level sensory areas. 31The mPFC is a crucial brain region involved in regulating higher-order functions like emotion, cognition, decision-making, and consciousness. 32A study found that delivery of cholinergic agonists into the mPFC during sevoflurane anesthesia can induce wakefulness, highlighting the mPFC's role in anesthesia regulation. 3Deactivating the mPFC delays the recovery of consciousness under general anesthesia. 33Specific excitation of the mPFC-dorsal medial thalamus circuit can reduce anesthesia depth and promote awakening, suggesting the mPFC's importance as a key downstream nucleus in the "Top-Down" regulation of general anesthesia. 34e PrL, a subregion of the mPFC, is closely connected to subcortical regions and plays a crucial role in cognitive functions and emotional regulation. 35,36However, its involvement in anesthesia-emergence processes has been understudied.In our previous study, we found that dopamine neurons in the VTA-PrL pathway exert a wake-promoting effect during sevoflurane anesthesia via the D1 receptor. 24One study reported that inactivation of PrL attenuates behavioral arousal induced by stimulation of the basal forebrain during sevoflurane anesthesia. 37 -VTA descending pathway as potential mediators in the transition between anesthesia induction and emergence, utilizing behavioral analysis, electroencephalographic recordings, and spectral analysis.Furthermore, we conducted microinjections of NMDA receptor antagonists (MK-801) or AMPA receptor antagonists (NBQX) into the VTA, aiming to elucidate the receptor mechanisms by which the PrL Pyr -VTA pathway regulates anesthesia and arousal.6J mice (8-10 weeks old), weighing 24-28 g, were obtained from the Experimental Animal Centre of the Chinese People's Liberation Army General Hospital.The mice were housed at the Experimental Animal Facility within the Anaesthesiology Department of the First Medical Centre, Chinese People's Liberation Army General Hospital.The mice were maintained at a room temperature of 22 ± 2°C under a 12 h light/dark cycle (lights on at 7:00 am), with free access to food and water.Behavioral tests were conducted between 09:00 and 18:00 Beijing time.All experimental procedures complied with the Regulations for the Management of Experimental Animals and Animal Experiments.Measurement of loss and recovery of righting reflex and electroencephalogram (EEG) monitoring were conducted 4-6 weeks post-viral injection.The Animal Ethics Committee approved the experimental protocol (SQ-2021139), which adhered to our research institution's guidelines for animal experiments.

| 3 of 14 CAO
coordinates were as follows: anterior-posterior (AP) +1.98 mm, medial-lateral (ML) ±0.25 mm, dorsal-ventral (DV) −2.1 mm.The total injection volume amounted to 120 nL.Following injection completion, the needle remained in place for 10 min before removal.A 200μm diameter ceramic ferrule was positioned 100 μm above the injection site following optogenetic virus injection.For PrL Pyr -VTA neural projection validation, either a microinjection cannula or a ceramic ferrule for optogenetic stimulation was implanted in the VTA.et al.Coordinates were AP −3.4 mm, ML ± 0.3 mm, and DV −4.0 mm.Upon completing the steps, three EEG electrodes were implanted in the skull.The coordinates for the three EEG electrodes were: Electrode 1: AP +1.0 mm, ML −2.0 mm; Electrode 2: AP −4.0 mm, ML −2.5 mm; and Electrode 3: AP −4.0 mm, ML +2.5 mm.Then, the electrodes, optical fibers, or microinjection cannulas were secured to the skull using diluted dental cement.
-VTA neural pathway, either the virus (rAAV2/9-DIO-hM3Dq-mCherry-WPRE-pA/rAAV2/9-DIO-hM4Di-mCherry-WPRE-pA) or the control virus (rAAV2/9-DIO-mCherry-WPRE-pA) was administered into the PrL region, and another virus (rAAV2/Retro-CaMKII-NLS-Cre) was injected into the VTA region.All viruses were supplied by OBiO Technology Co., Ltd.(Shanghai, China).Following viral injection, microinjection cannulas were implanted in the VTA region to investigate the mechanisms of action of the PrL Pyr -VTA neural pathway receptors.Behavioral tests were conducted 4 weeks after viral transduction and expression.Both the experimental and control groups of mice received an intraperitoneal injection of clozapine N-oxide (CNO) (MedChemExpress, USA) at a dose of 1 mg/kg.After 45 min, the mice were allowed to acclimatize in the righting reflex barrel for 15 min, followed by 2.0% sevoflurane inhalation anesthesia for 30 min.The durations of loss of righting reflex (LORR) and return of righting reflex (RORR) were documented.For receptor action investigation, DMSO, MK-801 (an NMDA receptor antagonist, 1.2 nmol/side), and NBQX (an AMPA receptor antagonist, 0.8 nmol/side) (MedChemExpress, USA) were administered via the cannulas.The LORR and RORR durations were recorded after 15 min of drug injection.

F I G U R E 3 3 . 6 |
Figure 5F, left), compared to the mCherry group.During the RORR process, there was an increase in the percentages of power in the δ bands in the hM4Di group (p < 0.001), while the percentages in the β and γ bands decreased (β, p = 0.0071; γ, p = 0.0111; Figure 5F, right) compared to the mCherry group.EEG spectral analysis revealed that inhibition of the PrL Pyr -VTA pathway led to an increase in activity in the low-frequency band associated with sleep and a decrease in activity in the high-frequency band associated with wakefulness.Following the completion of all behavioral experiments, the mice were euthanized and subjected to validation of virus expression.Widespread mCherry

F I G U R E 6
The PrL Pyr -VTA neural pathway exerts its effect mainly through NMDA receptors in VTA.(A) Schematic illustration of virus injection in the PrL and VTA regions, where the microinjection cannulas were implanted in the VTA region.(B) Left, righting reflex detection; right, the process of detecting anesthesia induction and emergence times through chemogenomic activation of the PrL Pyr -VTA neurons and blocking of NMDA/AMPA receptors in 2.0% sevoflurane anesthesia.(C) Representative brain slice with VTA microinjection cannula implantation.(D) Left, the mean anesthesia induction time is longer in the hM3Dq + DMSO group compared to mCherry+DMSO group (p < 0.001, n = 8), and the induction time is shorter in the hM3Dq + MK-801 group compared to the hM3Dq + DMSO group (p = 0.012, n = 8) (F (3, 28) = 14.66);Right, the emergence time is shorter in the hM3Dq + DMSO group compared to the mCherry + DMSO group (p < 0.001, n = 8), and the emergence time is longer in the hM3Dq + MK-801 group compared to the hM3Dq + DMSO group (p = 0.014, n = 8) (F (3, 20) = 8.373).Data are the mean ± SD; (*p < 0.05, **p < 0.01, and ### p < 0.001).